Immunization of C57BL/6 mice with GRA2 combined with MPL conferred partial immune protection against toxoplasma gondii

Background: We have previously reported that immunization with GRA2 antigen of Toxoplasma gondii induces protective immunity in CBA/J [H2k] and BALB/c mice [H2d]. We aimed to examine whether immunization of a distinct strain of rodent with recombinant dense granule antigens [GRA2] combined with monophosphorryl lipid A [MPL] adjuvant elicits protective immune response against T. gondii

Methods: C57BL/6 [H2b haplotype] mice were immunized with GRA2, formulated in MPL adjuvant

Results: Strong humoral response, predominantly of IgG1 subclass and cellular response, IFN-gamma, was detected at three weeks post immunization. Mice immunized with GRA2 had significantly [p < 0.01] fewer brain cysts than those in the adjuvant group, upon challenge infection. Despite the production of a strong antibody response, IFN-gamma production and brain cyst reduction were not significant when the immunized mice were infected four months after the immunization

Conclusions: We can conclude that GRA2 immunization partially protects against T. gondii infection in C57BL/6 mice, though the potency and longevity of this antigen as a standalone vaccine may vary in distinct genetic backgrounds. This observation further emphasizes the utility of GRA2 for incorporation into a multi-antigenic vaccine against T. gondii

Development of new recombinant DgK antigen for diagnosis of dirofilaria immitis infections in dogs using ELISA technique and its comparison to molecular methods

Background: Dirofilaria immitis is a cosmopolitan zoonotic, vector-borne parasite of carnivorous animals causing dirofilariasis in human beings. Common commercial serodiagnostic tests for canine dirofilariasis usually lead to different results in their sensitivity and specificity. The present study reports development of recombinant DgK [rDgK] antigen of D. immitis for accurate immunodiagnosis of D. Immitis-infected dogs using indirect ELISA test

Methods: The rDgK coding sequence was successfully sequenced, codon optimized and cloned in pET-24a[+] expression vector and then expressed in Escherichia coli. The recombinant DgK was affinity purified using Ni[+2] - charged HiTrap chelating column, followed by testing in Western blotting and enzyme-linked immunosorbent assays [ELISA] with dog sera from a dirofilariasis endemic area. The performance of rDgK ELISA was evaluated using 60 sera collected from suspected dogs, while molecular technique was used as a reference test

Results: Sera from positive control D. immitis infection produced a strong IgG antibody response to rDgK both in ELISA and Western blotting tests. The sensitivity and specificity related to diagnostic potential of rDgK for ELISA were 92.5% and 87.5%, respectively. The results of rDgK ELISA showed a high agreement [0.764] with molecular identification

Conclusions: The findings revealed that the developed new rDgK antigen is sensitive and specific for immunodiagnosis of canine dirofilariasis using ELISA test

Aluminium-induced oxidative stress, apoptosis and alterations in testicular tissue and sperm quality in wistar rats: ameliorative effects of curcumin

Background: Reproductive toxicity is a major challenge associated with aluminum [Al] exposure. No studies have evaluated the possible effects of curcumin [CUR] on Al-in- duced reproductive dysfunction. Therefore, this study investigated the effects of CUR treatment on Al-induced reproductive damage

Materials and Methods: In this experimental study, 40 male Wistar rats were allocated to the five groups [n=8] based on the treatment they received: no treatment [control], solvent [dimethyl sulfoxide [DMSO] or distilled water], CUR 10 mg/kg body weight [BW], Al chloride 10 mg/kg BW, and CUR+A1 chloride [10 mg/kg BW/each alone]. Treatments were performed by intraperitoneal [IP] injections for 28 days. The left testis was assessed for histopathological analysis as well as the incidence of germ cell apoptosis. One-way analysis of variance [ANOVA] followed by the Tukey's test was used. PO.05 was considered significant

Results: Significant reductions in body and testis weight; plasma testosterone and luteiniz-ing hormone levels; sperm count, motility, morphology, and viability; germinal epithelium thickness; seminiferous tubules diameter; as well as, superoxide dismutase activity were observed in rats treated with Al. Moreover, Al exposure caused significant increments in the lumen diameter of tubules, terminal deoxynucleotidyl transferase dUTP nick end labeling [TUNEL]-positive cells and malondialdehyde [MDA] levels compared to the control group. However, in rats receiving CUR+A1, CUR significantly reversed the adverse effects of Al on testis and sperm quality. No significant differences in follicle-stimulating hormone ' - . [FSH] levels and nuclear diameter of spermatogonia were detected among all groups

Conclusion: It can be concluded that Al causes reproductive dysfunction by creating oxi-dative damage. CUR, on the other hand, reduces the toxic effects of Al and improves the antioxidant status and sperm quality in male rats

Effects of crocin supplementation during in vitro maturation of mouse oocytes on giutathione synthesis and cytoplasmic maturation

Background: Crocin is an active ingredient of saffron [Crocus sativus L] and its an-tioxidant properties have been previously investigated. This carotenoid scavenges free radicals and stimulates glutathione [GSH] synthesis; consequently, it may protect cells against oxidative stress. The aim of this research is to protect oocytes from oxidative stress by the addition of a natural source antioxidant

Materials and Methods: In the present in vitro experimental study, we collected cumulus oocyte complexes [COCs] from mouse ovaries of euthanized, 6-8 week-old female Naval Medical Research Institute [NMRI] mice. Oocytes were subjected to in vitro maturation [IVM] in the presence of either crocin [5 or 10 microg/ml], 5 mM buthionine-[S-R]-sulfoximine [BSO], or the combination of crocin plus BSO. Oocytes that matured in vitro in a medium without crocin or BSO supplements were considered as controls. Following 16-18 hours of IVM, matured oocytes [n=631] were fertilized by capacitated sperm from NMRI male mice, and cultured in vitro for up to 96 hours to assess preimplantation embryonic development. The levels of GSH in metaphase II [Mil] oocytes after IVM [n=240] were also assessed by the 5, 5-dithio-bis [2-nitrobenzoic acid] [DTNB]-GSH reductase recycling assay

Results: Supplementation of IVM media with 10 microg/ml crocin significantly [P<0.05] increased nuclear maturation, preimplantation development and GSH concentrations compared with the control group. Maturation of oocytes in IVM medium supplemented with BSO alone or the combination of microg/ml crocin and BSO drastically decreased GSH concentrations and subsequently resulted in low rates of maturation, fertilization and blastocyst development. However, the combination of 10 microg/ml crocin with 5 mM BSO increased the level of nuclear maturation which was comparable to the control group

Conclusion: Supplementation of IVM media with crocin can improve nuclear maturation rates and subsequent developmental potential of mouse oocytes. This may occur by its beneficial effect in increasing GSH concentrations in Mil oocytes

The relationship between depression and self-care in patients with type II diabetes

Depression is a common situation in patients with diabetes that may reduce the ability of individuals to engage in self-care behaviors. The present study was performed to investigate the relationship between depression and self-care in patients with type II diabetes. This descriptive, cross-sectional study was performed with convenience sampling on 200 patients with type II diabetes who were referred to Saqqez Imam Khomeini Hospital, Sanandaj, Iran, in 2012. The data collection instrument consisted of three demographic information, depression, and self-care questionnaires. Data were analyzed by SPSS for Windows [version 16; SPSS Inc., Chicago, IL, USA] using chi-square and descriptive statistics. P values less than 0.05 were considered statistically significant. Diet in 5-7 days a week [P = 0.002] and adherence in 5-7 days a week [P = 0.03] were significantly lower in patients with diabetes and depression than in diabetic patients without depression. In the areas of exercise, foot examination, and self-monitoring of blood glucose there was no significant difference between the two groups. Given the high prevalence of depression in diabetic patients, using screening methods such as HANDS scale seems to be necessary for disorder identification and promoting self-care activities

Hydroxyl capped silver-gold alloy nanoparticles: characterization and their combination effect with different antibiotics against Staphylococcus aureus

Metal nanoparticles [NPs] offer a wide variety of potential applications in pharmaceutical sciences due to the unique advances in nanotechnology research. In this work, bimetal Ag-Au alloy NPs were prepared and their combinations with other antibiotics were tested against Staphylococcus aureus. Firstly, Ag-Au alloy NPs with Au/Ag molar ratio of 1:1 was fabricated and was purified by agarose gel electrophoresis system. The morphology and size of the purified NPs were confirmed by transmission electron microscopy. Chemical composition and surface chemistry of these NPs were studied with atomic absorption spectophotometry and Fourier transforms infrared spectroscopy, respectively. The size of purified Ag-Au alloy NPs was less than 200 nm. Also the presence of organic compounds with a hydroxyl residue was detected on the surface of these purified NPs. In next step the effect of purified Ag-Au alloy NPs on the antibacterial activity of different antibiotics was evaluated at sub-inhibitory content [5 microg/disk] using disk diffusion method against S. aureus. Ag NPs and Au NPs were also tested at same content [5 microg] using mentioned method. The most enhancing effect of Ag-Au alloy NPs was observed for penicillin G and piperacillin. No enhancing effects on the antibacterial activity of different antibiotics were observed at 5 microg/disk for the mono-metal nanoparticles [Ag NPs and Au NPs] against S. aureus. These results signify that the Ag-Au alloy NPs potentiates the antimicrobial action of certain antibiotics suggesting a possible utilization of this nano material in combination therapy against resistant S. aureus

IgG AVIDITY WESTERN BLOT USING Toxoplasma gondii rGRA-7 CLONED FROM NUCLEOTIDES 39-711 FOR SERODIAGNOSIS OF ACUTE TOXOPLASMOSIS / Avidez do IgG pelo Western Blot usando o r GRA-7 do Toxoplasma gondii clonado de nucleotídeos 39-711 para sorodiagnóstico de toxoplasmose aguda

Toxoplasmosis is an important cause of congenital infection. The present study was performed to evaluate the usefulness of recombinant (r) GRA-7 cloned from nucleotides (n) 39-711 in discriminating between acute and chronic toxoplasmosis. First, commercial IgM, IgG and IgG avidity ELISAs were used to determine the serological profile of the sera. Serum samples were from 20 symptomatic patients with acute infection (low IgG avidity, IgM positive), 10 with chronic infection (high IgG avidity, IgM negative) and 10 with indeterminate IgG avidity (IgM positive) which were tested for IgG avidity status with an in-house developed IgG avidity Western blot using the rGRA-7 recombinant antigen. All 20 sera from cases of probable acute infection showed bands which either faded out completely or reduced significantly in intensity after treatment with 8 M urea, whereas the band intensities of the 10 serum samples from chronic cases remained the same. Of the 10 sera with indeterminate IgG avidity status, after treatment with 8 M urea the band intensities with six sera remained the same, two sera had completely faded bands and another two sera had significantly reduced band intensities. Discrimination between acute and chronic toxoplasmosis was successfully performed by the in-house IgG avidity Western blot.

Toxoplasmose é uma causa importante de infecção congênita. O presente estudo foi feito para avaliar o uso do recombinante (r) GRA-7 clonado de nucleotídeos (n) 30-711 para discriminar entre toxoplasmose aguda e crônica. Inicialmente IgM, IgG e ELISA avidez IgG comerciais foram usados para determinar o perfil sorológico do soro. Amostras de soro de 20 pacientes sintomáticos com infecção aguda (IgG avidez baixa, IgM positivo), 10 com infecção crônica (alta avidez IgG, IgM negativo) e 10 com avidez IgG indeterminada (IgM positivo) que foram testados para o status de avidez IgG com um doméstico Western Blot desenvolvendo avidez IgG usando o rGRA-7 antígeno recombinante. Todos os 20 soros de provável infecção aguda mostraram bandas que ou se apagaram completamente ou tiveram a sua intensidade significantemente reduzida após tratamento com uréia 8 M, enquanto as intensidades das bandas das 10 amostras de soros de casos crônicos permaneceram iguais. Dos 10 soros com status indeterminado de avidez de IgG, após tratamento com uréia 8 M a intensidade das bandas em seis soros permaneceram iguais, dois soros tiveram bandas apagadas completamente e dois outros tiveram significante redução da intensidade das bandas. Discriminação entre toxoplasmose aguda e crônica foi feita com sucesso através do IgG avidez Western blot doméstico.

In vitro maturation, fertilization and embryo culture of oocytes obtained from vitrified auto-transplanted mouse ovary

The purpose of this study was to investigate the in vitro survival and developmental potential of oocytes obtained from vitrified mouse ovaries transplanted to a heterotopic site. In this experimental study, two-week-old mice were unilaterally ovariectomized after anesthesia. The ovaries were vitrified by cryotop. After two weeks, the ovaries were thawed and autotransplanted to the gluteus muscle tissue. Three weeks later the mice were killed, after which we removed and dissected the transplanted and opposite right ovaries. Cumulus oocyte complexes [COCs] and denuded oocytes were evaluated for in vitro maturation [IVM], in vitro fertilization [IVF] and in vitro development [IVD]. The control group consisted of sevenweek- old age-matched mice ovaries. All vitrified-transplanted [Vit-trans] ovaries contained some oocytes that survived. Following IVM, IVF and IVD, there were 41.7% out of 12 cultured zygotes that reached the 8-cell stage. Our experiment supports the progressive role of long-term graft survival after wholeovarian cryopreservation by vitrification and subsequent heterotopic transplantation. It is possible to recover viable follicles and oocytes that have the ability to develop in vitro

[Effect of incorporation of various amounts of nano-sized hydroxyapatite on the mechanical properties of a resin modified glass ionomer]

Nano-sized hydroxyapatite nano particles [nHA] have optimal biological properties and by incorporating them into the restorative materials, we can benefit from these properties. The present study sought to assess the effect of incorporation of various amounts of nHA on the mechanical properties [compressive and flexural strengths] of a resin modified glass ionomer. In this experimental study, a total of 252Fuji II LC improved GI samples were divided into 6 groups including a control group [0%] and the nHA groups [1%, 2%, 5%, 7% and 10% based on mass percent]. Of the samples, 108 were fabricated for the flexural strength testing using a two-piece aluminum mold [2x2x25 mm] according to ISO standard 4049. In each group, a total of 18 samples in 3 subgroups [6 samples each] were fabricated. All samples were removed from the mold after 80 seconds of light irradiation. A total of 144 samples made for compressive strength testing using a two-piece brass mold [4x6 mm] according to ISO standard 9917. Thus, in each group, a total of 24 samples in three subgroups [8 samples each] were fabricated and removed from the mold after 80 seconds of light irradiation. After removal from the mold, all samples were stored in an incubator at 37°C and 100% moisture and underwent flexural and compressive strength testing with the primary load of 2 N and crosshead speed of 0.5 mm/min[-1] in a Zwick testing machine after one day, one week and one month. Data were analyzed using normal statistical tests of one-way ANOVA and Tukey's HSD [Post Hoc]. In order to assess the correlation of time [in groups with various mass percentages of HAP] with understudy outcomes, one way ANOVA was employed and P<0.05 was considered statistically significant. Study results showed that incorporation of 5% nHA resulted in a significant increase in flexural strength after 30 days [17.4 MPa increase][P<0.05]. Also, if the nHA weight percent exceeded 5%, flexural strength suffered a dramatic drop after one month[28.9 MPa reduction]. This study also showed that addition of 5% NHAP significantly increased flexural elastic modulus after one month [2.2 GPa increase]. Addition of NHAP to resin modified glass ionomer caused a small increase in compressive strength and compressive elastic modulus which was not statistically significant [about 1MPa increase]. Addition of nHA to resin modified glass ionomers [Fuji II LC improved] not only does not reduce compressive strength, but also can increase flexural strength which would be the greatest if nHA is added in an amount of 5%

Analysis of relation between C677T genotype in MTHFR gene and prostatic cancer in Iranian males

Methylenetetrahydrofolate reductase [MTHFR] enzyme is one of the most important enzymes with a pivotal role in the folate metabolism and DNA synthesis pathways. Single nucleotide polymorphism [SNPs] in the coding gene has been related to many medical diseases as well as diverse malignancies including the prostate cancer which is the leading cause of the cancer deaths in men and one of the major public health problems. The goal of this study is to determine the relationship between the MTHFR C677T SNP and the prostate adenocarcinoma in Iranian males attending to the Labbafi-nezhad hospital in Tehran. In this Case-control unmatched study, 67 and 75 paraffinized tissue samples were taken out of the specimens diagnosed previously as the prostatic adenocarcinoma and nodular prostatic hyperplasia for the case and control groups respectively. MTHFR C677T genotyping was done by the use of multiplex ARMS-PCR and frequencies of the alleles were compared between the case and control groups as well as calculating the deviation from Hardy-Weinberg equilibrium and Odds Ratio for the "T" allele regarding the prostatic carcinoma. The observed rates in the control group were not too different from that of expected from Hardy- Weinberg equilibrium [P=0.407]. Frequencies of the possible genotypes were as follows: CC, 43.28% vs. 42.67%; CT, 49.25% vs. 52% and CT, 7.46% vs. 5.33% in the case and control groups respectively [P=0.85]. 1.37 times increased risk was found for the homozygote carriers of C677T variant [OR: 1.37, 95% CI: 0.33- 5.6; P=0.653] which is however statistically not significant. No association has been evident between the MTHFR 677C>T polymorphism and the risk of prostatic carcinoma in this study confirming the findings of some of the previous attempts; however, [OR: 1.37, 95% CI: 0.33-5.6] implies a slight effect of the homozygote on the carcinogenesis. Thus larger studies especially with a greater number of the smaples are recommended

Functional Recombinant Extra Membrane Loop of Human CD20, an Alternative of the Full Length CD20 Antigen

Targeting of CD20 antigen with monoclonal antibodies has become the mainstay in the treatment of non-Hodgkin's lymphomas and immunotherapeutic depletion of malignant B cells. Accessibility of antigen is one of the crucial factors in development of monoclonal antibodies against this antigen. One major problem in expression of full length CD20 is aggregation and misfolding. Therefore, production of an alternative polypeptide is easer and favorable comparing to that of a full length transmembrane protein CD20. In this study, we expressed the extra membrane loop of hCD20 [exCD20] consisting of a non-glycosylated 47-amino acids region. The exCD20 coding sequence was amplified by PCR and cloned in pET32a[+] expression vector. The desired protein was expressed in fusion with thioredoxin and 6 His tag in E. coll Origami strain. ELISA and Western-blotting data were performed to indicate the functionality of this protein. We have obtained the exCD20 recombinant protein which can be detected in ELISA and Western-blot experiments. This recombinant fusion protein was soluble and stable without aggregation and misfolding problems. Conclusion: The recombinant extra membrane loop of human CD20 protein in fusion with thioredoxin [exCD20] can be used in function assays and some applications such as ELISA, immuneblotting, affinity purification, immunization, screening, and development of anti-CD20 antibodies

[Effect of interleukin -27 on recovery from experimental autoimmune encephalomyelitis in C57BL/6 mice]

Multiple sclerosis and its murine model, experimental autoimmune encephalomyelitis [EAE], are chronic inflammatory demyelinating diseases of CNS. This study aimed to examine the effects of IL-27coding plasmid on disease status and certain immunological parameters in EAE-affected C57BL/6 mice. IL-27 gene was subcloned in P240 plasmid. The recombinant P240-mIL27 and P240 plasmids were injected two times, each time 200 micrograms, to test and control EAE mice, respectively. The clinical signs of the treated mice were evaluated daily and scored according to a standard method. One week after the last injection, all mice were sacrificed. The ELISA and MTT tests were performed to evaluate the production of IL-4, IFN-? and IL-17 from and proliferative response of splenocytes against specific antigen challenge, respectively. Furthermore, to demonstrate the immune cells infiltration, histopathological exam was performed on the brainstem of mice. The P240-mIL27 plasmid could significantly improve clinical course of EAE in the test group. Also in this group, the level of IL-4 was greater than that in the control group, while the levels of IFN-? and IL-17 were lower than those of the control group. In MTT test, the splenocytes of the test group showed a significantly less proliferative response than the control group. Finally, less infiltration of immune cells was seen in the brainstem of EAE mice treated with P240-mIL27 plasmid. IL-27 by shifting the immune responses from inflammatory Th1/Th17 towards anti-inflammatory Th2-type responses could be a suitable candidate for the treatment of inflammatory diseases such as MS

Expression and single-step purification of GRA8 antigen of toxoplasma gondii in Escherichia coli

Diagnosis of Toxoplasma gondii [T.gondii] infection is of great medical importance especially for pregnant women and immunosuppressed patients. Numerous studies have shown that the recombinant production of several tToxoplasma antigens, including dense granule antigens [GRAs] has a great potential as diagnostic reagents. Previous studies reported expression of amino terminal GRA8 protein in fusion with large tags. In the present study, we produced truncated GRA8 [GRA8], excluded from the signal peptide and C-terminal transmembrane domain, with a short fusion tag in Escherichia coli. [E.coli]. GRA8 was purified using an optimized single-step Iimmobilized Mmetal ion Aaffinity Cchromatography [IMAC]. The purity and yield of GRA8 was highest at pH= 9.25. At this pH, 13.6 mg of GRA8 was obtained with the purity of 97.97%. Immunogenicity of the protein was evaluated in Western blot analysis showing the serum sample from a rabbit immunized with GRA8 recognized a single antigen of T. gondii tachyzoite at the expected molecular weight of native GRA8. To diagnosis acute tToxoplasma infection in pregnant women, an indirect immunoglobulin M [IgM] enzyme-linked immunosorbent assay [ELISA] was developed using GRA8 resulting in a test specificity and sensitivity of 97.1% and 60.6%, respectively. These results demonstrated that immunogenic GRA8 can be produced in fusion with a short tag and purified near to homogeneity using an optimized IMAC. GRA8-IgM-ELISA was useful for detection of acute toxoplasma infection

Construction and in vitro expression analyses of a DNA plasmid encoding dense granule gra5 antigen of toxoplasma gondii

Toxoplasmosis is an infection caused by the protozoan parasite Toxoplasma gondii [T.gondii] throughout the world. Although usually asymptomatic, the infection can cause serious medical problems in immunocompromised individuals and fetuses. Toxoplasmosis also causes considerable economic loss because of abortion in livestock. DNA vaccination is a promising approach against intracellular parasites such as T.gondii. The goal of this study was to construct and evaluate functionality of a mammalian plasmid expressing GRA5 antigen of T.gondii as a possible DNA vaccine. GRA5 gene fragment devoid of the signal sequence, was amplified from genomic DNA of T.gondii RH strain, and cloned into pcDNA3.1 plasmid. The pcDNA3.1-GRA5 [pGRA5] was analyzed by restriction enzyme digestion followed by sequence determination. The pGRA5 was transfected into HEK 239-T human kidney cells, and expression of GRA5 antigen was investigated by Western blotting and immunofluorescence staining. The sequence encoding GRA5 was cloned into pcDNA3.1 plasmid. Restriction digestion of pGRA5 with Pst I enzyme showed correct in-sertion of GRA5 DNA into the plasmid. Sequence analysis revealed 100% homology with the published sequence of gra5. immunofluorescence and Western blotting analyses of HEK 293-T cells transfected with pGRA5 showed specific expression of GRA5. Immunogenicity of pGRA5 will be evaluated in mice

Production of recombinant human T lymphotropic virus type 1 tax protein in Rosetta [DE3] bacterial host

HTLV1 is the first detected retrovirus causing disease in human. The physiopathology of HTLV1 related diseases was mainly linked with its Tax protein characteristics. Use of mutant Tax proteins accompanied by immune regulator drugs could help treating HTLV1 associated myelopathy patients as a modulator of potent immune response against this viral protein. Since Tax protein is not commercially available, production of recombinant Tax protein was targeted for this study. Coding sequence of Tax protein [containing R222K mutation] in the pcDNA3.1[+] was digested with BamHI and XhoI restriction enzymes, and then removed and inserted into the expression vector pET32a[+] within the same cutting sites and cloned into E.coli DH5alpha. Recombinant vector was confirmed with enzymatic digestion, colony PCR, and sequencing of cloned gene. E.coli Rosetta [DE3] was transformed with the recombinant plasmid and the expression was induced. The expression of protein was assayed with SDS-PAGE and western blot using monoclonal antibodies against Tax and 5His epitope. Finally, antigenic characteristic of the recombinant protein was evaluated by western blotting against patient sera. Presence of Tax protein band in the SDS-PAGE and western blot was confirmed. Western blotting of the recombinant protein with patient sera showed the band related to Tax protein. The recombinant protein is well produced and could be detected by patients' sera, making it eligible to be used as a recombinant viral antigen for future purposes

Bacterial production of dense granule antigen GRA8 of Toxoplasma gondii

Dense granule antigens [GRA antigens] of Toxoplasma gondii induce strong antibody response in humans and are considered as useful diagnostic antigens. Previous studies reported expression of amino terminal GRA8 protein in fusion with large tags such as glutathione-S-transferase. The present study aimed to produce soluble full length immunogenic GRA8 in bacteria. GRA8 complementary DNA [cDNA], encoding amino acids 24 to 258, was amplified from tachyzoites of RH strain and cloned in prokaryotic expression vector pET-28b[+]. Expression of recombinant GRA8 [rGRA8] was analyzed by SDS-PAGE. Antigenicity and immunogenicity of the protein were evaluated by Western-blotting. The cloned gene fragment exhibited complete similarity with the published sequence of GRA8 gene by sequence analysis. rGRAS was expressed in Escherichia coli infusion with a very small tag and the soluble protein was purified by immobilized metal ion affinity chromatography. In immunoblot, serum sample from a rabbit immunized with rGRAS recognized a single antigen of T. gondii tachyzoite at the expected molecular weight of native GRA8. Sera from acutely-infected pregnant women strongly reacted with rGRAS in Western-blotting, while sera from chronically-infected or T. gondii-negative women failed to recognize the protein. The full length soluble rGRAS was successfully produced in E. coli and shown to be a highly immunogenic protein. As a result it could be used in immunological as well as molecular biology experiments

Deletions in the survival motor neuron gene in Iranian patients with spinal muscular atrophy

<p><b>INTRODUCTION</b>Spinal muscular atrophy (SMA) is a common neuromuscular disorder with progressive paralysis caused by the loss of alpha-motor neurons in the spinal cord. The survival motor neuron (SMN) protein is encoded by 2 genes, SMN1 and SMN2. The most frequent mutation is the biallelic deletion of exon 7 of the SMN1 gene. In SMA, SMN2 cannot compensate for the loss of SMN1, due to the exclusion of exon 7. The aim of our study was to estimate the frequency of the common SMN1 exon 7 deletion in patients referred to our centre for carrier detection and prenatal diagnosis.</p><p><b>MATERIALS AND METHODS</b>We performed the detection of exon 7 deletion of the SMN1 gene for the affected patients and fetuses suspected to have SMA.</p><p><b>RESULTS</b>Of 243 families, 195 were classified as SMA type I, 30 as type II, and 18 as type III according to their family histories. The analysis of exon 7 deletion among living affected children showed that 94% of the patients with SMA type I, 95% with type II families and 100% with type III had homozygous deletions. Of the prenatal diagnoses, 21 (22.8%) of the 92 fetuses were found to be affected and these pregnancies were terminated.</p><p><b>CONCLUSIONS</b>The homozygosity frequency for the deletion of SMN1 exon 7 for all 3 types was (94%), similar to those of Western Europe, China, Japan and Kuwait.</p>

Vancomycin use in a large teaching hospital in Shiraz, Islamic Republic of Iran, 2003

We investigated adherence to the Hospital Infection Control Practice Advisory Committee [HICPAC] guidelines on vancomycin prescription in a large university-affiliated hospital in Shiraz. From August to December 2003, 200 hospitalized patients received vancomycin. For only 12 [6%] of these patients was vancomycin prescribed appropriately according to HICPAC guidelines. The main reasons why vancomycin use did not comply with HICPAC recommendations were: surgical prophylaxis in patients with negative cultures for resistant Gram-positive organisms, no investigation of vancomycin serum levels in patients receiving > 48 hours of vancomycin, vancomycin serum levels not repeated in patients receiving > 1 week of vancomycin, no appropriate adjustment of dosage with respect to serum levels in patients receiving vancomycin

[Surveying SMN gene deletions in Iranian patients with spinal muscular atrophy and prenatal diagnosis]

Spinal muscular atrophy is a group of alpha-motor neuron. There are three genes for this disorder, of which SMN with two copies centromeric and telomeric is the most important one. In 95% of SMA patient's telomeric copy of SMN is homozygously deleted and the remaining has point mutation in this gene. In most of the patient's, exon 7 and 8 of SMN 1 is deleted. Therefore, analysis of SMN1 mutation is very important for carrier detection. The aim of this study was analysis of SMN1 mutation and determination of its frequency among Iranian patients. After genetic counseling and estimation of clinical symptoms of patients based on SMA consortium, molecular analysis based on PCR-RFLP has been performed. Frequency of consanguineous marriage in our study was 60%, while most of the patients were come from central and northern part of Iran. Of 243 families, 195 were categorized as type I, 30 as type II, and 18 as type III. Analysis of exon 7 deletion among families with live affected child showed that 94% of families with SMA type I, 95% in type II families and 100% in SMA type III had homozygous deletion. In prenatal diagnosis, twenty one of ninety two [22.8%] fetal samples were found to be affected and these pregnancies were terminated. The frequency of homozygous deletion of exon 7 of SMN1 was 94%. This is in agreement with Western Europe, China, Japan, and Kuwait

[Functional iron deficiency by percentage of hypochromic red cell in patients treated by chronic hemodialysis in Ghaem and Emam Reza hospital in 2004-2005]

The availability of recombinant human erythropoietin [rHuEPO] over the past decade has advanced the management of anemia in hemodialysis patients. However, iron deficiency either absolute or functional may be leading to failure of rHuEPO treatment. Serum Ferritin [SF] and percent of Transferrin saturation [TSAT] are regarded as the preferred indirect measurements of iron status. These tests, however, have practical limitations and lack sensitivity and specificity to identify ''functional iron deficiency'', witch can occur in the presence of normal or even increased iron stores. Newer methods of assessing iron status are available; with a percentage of hypochromic red cells [%Hypo] >2.5 have been proposed as a marker of Iron deficient erythropoiesis. It measured Hematocrit, Hemoglobin content, SF, and TSAT over 76 chronic hemodialysis patients. The percentage of hypochromic red cells was also measured in a subset of 39 patients. Functional Iron deficiency was defined as a SF > 100 micro g/1 and TSAT 8%. Indivisual characterizing and result of 1ab collected by questinnaire and analyzed by descriptive statistics. 13% of our patients had serum ferritin values > 100 micro g/1 and transferrin saturaton 8% in 80% of our patients at this group. It observed a significant difference in Transferrin saturation [p=0.000] and %Hypo [p=0.001] in these two groups. The diagnostic sensitivity and specificity of %Hypo > 8% in detecting functional Iron deficiency were 80% and 79% respectively. In hemodialysis patients on rHuEPO therapy, Hypo > 8% is a sensitive available marker to identify functional iron deficient patients